肺静脉狭窄上游血管EndMT的TGF-β调控机制研究

Pulmonary vein stenosis remains a challenging congenital heart disease. We previously established a piglet pulmonary vein stenosis model, and found significant intimal hyperplasia in the upstream pulmonary vein, as well as increased tissue transforming growth factor-β (TGF-β) level. Endothelial-to-mesenchymal transition (EndMT) might be involved in this process. Post-operative administration of Losartan reduced the intimal hyperplasia, reversed the reduction of VE-Cadherin, and improved hemodynamics, but the underlying mechanisms are unknown. A recent study from Harvard University found that Losartan suppressed TGF-β mediated EndMT through non-canonical pathway (p-ERK) in mitral valve endothelial cells; however, the controlled endothelial cells from carotid artery didn’t undergo EndMT, indicating EndMT is specific to cellular source. To our knowledge, whether pulmonary vein endothelial cells have the feature of EndMT is unclear so far and needs further investigation. Therefore, we hypothesize that upstream pulmonary vein undergoes EndMT change triggered by the elevated focal TGF-β level, and EndMT activates downstream Snail gene, then inhibits the expression of VE-Cadherin. Losartan may block this pathway, and ameliorate EndMT, therefore reduce intimal hyperplasia, probably through the non-canonical pathway of TGF-β. In addition to our previous experiences, the present study will investigate the EndMT related cellular/proteinic/genetic alternations in the upstream pulmonary vein in a piglet model of pulmonary vein stenosis. Pulmonary vein endothelial cells are isolated from the upstream vessels beyond the stenosis and cultured in vitro within an environment mimicking high TGF-β concentration and high sheer stress, with or without inhibitors against different steps of TGF-β pathway. By performing both in vivo and in vitro studies, the underlying EndMT mechanism is to be elucidated in the pulmonary vein stenosis, as well as the regulatory mechanism of TGF-β pathway on EndMT. This study also aims to explain the underlying mechanism of potential therapeutic effect of Losartan (probably TGF-β related) and provides new evidences for treatment of pulmonary vein stenosis.

肺静脉狭窄仍是先天性心脏病的治疗难题。前期研究建立猪肺静脉狭窄模型，发现狭窄上游血管内膜显著增生，局部TGF-β表达升高，内皮间质转化（EndMT）可能参与这一过程；术后口服氯沙坦可减轻内膜增生，逆转VE-Cadherin的下降，但机制不清。最近一篇研究发现氯沙坦通过抑制非经典途径阻断TGF-β诱导的心脏瓣膜内皮EndMT进程，我们推测狭窄上游肺静脉局部TGF-β表达升高激活EndMT进程，下游snail基因激活后抑制VE-Cadherin表达；应用氯沙坦干预可能阻断该通路，减缓EndMT，减轻内膜下增生。本课题拟在前期基础上，应用肺静脉狭窄模型，检测上游肺静脉内膜EndMT相关细胞、蛋白和基因表达；培养上游肺静脉内皮细胞，模拟体内高TGF-β状态，在TGF-β信号通路的多个环节进行干预对比，重点探讨TGF-β调控EndMT的机制，并阐述氯沙坦的作用原理，为临床治疗肺静脉狭窄提供新依据。

儿童肺静脉狭窄缺乏有效的治疗手段，无论是支架还是外科治疗效果均不佳，尤其是弥漫性狭窄，仍是先天性心脏病的治疗难题。本研究在前期基础上，改良了幼猪肺静脉狭窄模型，仅应用单次左侧开胸，初始环缩程度较既往模型更严重；术后1月复查心超可见连续性肺静脉血流频谱，伴平均压差升高，确认狭窄形成，术后2月处死动物获取组织标本时可见环缩处完整、管腔变窄，再次确认了狭窄模型的可靠性。根据预订计划分为假手术组、肺静脉环缩组和氯沙坦处理组，动物处死前三组之间血流动力学差异显著，环缩组右心室压力显著升高，而氯沙坦处理组可部分降低该效应。HE染色证实狭窄上游肺静脉血管内膜显著增生，血管内皮细胞极性、形态发生改变，CD31、α-SMA免疫荧光共染色也进一步证实以上发现，符合内皮间质细胞转换（EndMT）的特征。狭窄组上游肺静脉组织行Western blotting检测提示YAP、TAZ蛋白表达在环缩的上游静脉显著上调，未环缩的右中肺静脉中YAP、TAZ蛋白表达无明显变化；氯沙坦可部分降低环缩引起的YAP、TAZ改变效应。体外实验中，培养人脐静脉血管内皮细胞（HUVEC），发现加入氯沙坦可在0.5小时、4小时显著抑制YAP、TAZ蛋白的表达，与在体实验结果呈类似趋势，提示YAP/TAZ相关的Hippo通路可能在肺静脉狭窄以及氯沙坦的保护作用中发挥了重要的作用。本课题的后续研究将进一步阐明其中的具体上下游机制，并利用已成功构建的自体肺静脉内皮细胞原代培养进一步验证，期望为临床应用氯沙坦防治肺静脉狭窄提供理论基础。